Does Charge Repulsion of Amyloids Regulate their Assembly Pathways and Toxicity? Deposits of amyloid fibrils, insoluble protein fibrils with cross beta-sheet structure, are characteristic of numerous human disorders, including Alzheimer's disease, Parkinson's diseases and type II diabetes. Research over the past decade has indicated that oligomeric intermediates, transiently formed during fibril growth, represent the main molecular species harmful to tissues. This proposal will investigate how the net strength of intermolecular and intramolecular protein interactions regulates amyloid fibril assembly. The particular focus will be on repulsive charge interactions and their potential role in selecting assembly pathways, the structure of various intermediates and their resulting cellular toxicity. We will use two distinct, medically relevant and natively folded amyloid proteins: lysozyme and b-2 microglobulin. Experimentally, our approach relies on an integrated use of a wide variety of techniques, including static and dynamic light scattering, atomic force microscopy, as well as fluorescence, CD and FTIR spectroscopy. The proposal has three specific aims relating to these questions. Experiments in aim 1 will ascertain whether the transition from net repulsion to attraction among amyloidogenic monomers under various solution conditions predicts the transition from fibril formation to precipitation. The intriguing corollary to this aim is that net repulsion is a prerequisite of ordered fibril assembly. In aim 2 we will use in situ dynamic light scattering and correlated atomic force microscopy to confirm whether fibril assembly can always follow distinct assembly pathways, not all of which involve the formation of oligomeric intermediates. We will further test whether transitions among different pathways are related to the effects of charge stress on both monomer structure and the structure of early intermediates. Aim 3 targets the relationship between cellular toxicity and the structure of oligomeric vs. non- oligomeric intermediates from different assembly pathways. In particular, we will determine the internal secondary structure of oligomeric vs. non-oligomeric intermediates along different pathways. The cellular toxicity of the same intermediates will be determined using a cellular toxicity assay. These experiments should provide basic insights into the structure-dysfunction relationship of various intermediates emerging along different fibril assembly pathways. 2.